Apparatus for tissue preparation

ABSTRACT

A tissue-embedding assembly is shown which is convertible to a resin option from a paraffin option. The tissue-embedding assembly permits the simultaneous embedding of multiple-tissue specimens with degassed embedding material. Full observation of the process as well as manipulation of the specimens is available during and up to solidification of the degassed embedding material. A novel heat transfer arrangement is available which permits the heating of paraffin to cause melting and embedding without disconnection of the refrigeration.

United States Patent Adams 1 Feb. 8, 1972 [54] APPARATUS FOR TISSUEPREPARATION [72] inventor: Robert P. Adams, Walden, NY.

[73] Assignee: Cenco Medical/Health Supply Corporation, Chicago, Ill.

[22] Filed: Apr. 16, 1970 21 Appl. No.: 29,128

[56] References Cited UNITED STATES PATENTS 3,009,258 ll/l96l Taylor..34/92 2,803,888 8/1957 Cerletti...

1,884,429 10/1932 Warner ..34/5X ii i l g FOREIGN PATENTS ORAPPLICATIONS 932,043 7/1963 Great Britain ..34/5

Primary Examiner-Charles Sukalo Assistant Examiner-W. C. AndersonAttorney-Robert E. Wagner [57] ABSTRACT A tissue-embedding assembly isshown which is convertible to a resin option from a paralfin option. Thetissue-embedding assembly permits the simultaneous embedding ofmultiple-tissue specimens with degassed embedding material. Fullobservation of the process as well as manipulation of the specimens isavailable during and up to solidification of the degassed embeddingmaterial. A novel heat transfer arrangement is available which permitsthe heating of paraffin to cause melting and embedding withoutdisconnection of the refrigeration.

5 Claims, 6 Drawing Figures PATENTH] FEB 8 I972 7 T; .1. souRcE SHEET 1BF 2 INVEN TOR ROBERT P ADAMS BY Ci ay A I ATT Y PATENTEU FEB 8 I972SHEET 2 [1F 2 INVENTOR ROBERT P ADAMS APPARATUS FOR TISSUE PREPARATIONThis invention relates to improvements in tissue preparation and, morespecifically, is directed to a new and improved apparatus for tissuedrying and embedding as well as a novel method for performing tissueembedding.

A great deal can be learned from a pathologistsexamination of a smallportion of tissue. In order to effectively carry out such examination,the orientation of the cells in the tissue sample should be undisturbed.This is accomplished through drying the specimen and then filling thevoids or spaces between the fibers or cells with liquid paraffin orresin which is allowed to solidify, usually through cooling. Thereafter,the embedded tissue is cut into thin section to permit microscopicexamination and other study, including such subjects as histology,arasitology, bacteriology, botany, zoology and kindred subjects. Suchtissue study techniques of this general type have been employed in thepast with considerable success, however, certain areas requireimprovement if effectiveness of this type of examination is to beincreased.

Generally, in the past, the process has included the selection of aproper tissue, dehydration with one of several dehydrants, clearing ofthe tissue with a soluble oil or acceptable clearing agent andthereafter infiltration of the tissue with the paraffin wax orcombination of wax and resinous material which is thereafter allowed toharden. An instrument commonly known as a microtome" is used to slicethin sheets of the impregnated tissue in the block of paraffin so thatit may be used on a slide for microscopic study.

Problems have been encountered in the past with prior art designsbecause of the poor visibility during drying and embedding afforded byprior art dryers. Poor heat transfer between the cooling or heatingmedium and the specimen was an added problem which was brought about bythe type of support for the tissue. Allied problems were encountered inthat when the specimen was required to be manipulated during theembedding step, only one specimen could be embedded at a time. Smallspecimens were oftentimes apt to be lost or damaged because of thenature of the drying and embedding operation.

The present invention overcomes the difficulties encountered in theprior art through the provision of a new and improved apparatus fortissue preparation which is of greatly simplified construction. Thepresent design uses a known and versatile type of refrigeration andvacuum source, having an upstanding evaporator coil which is in intimatetransfer contact with a support structure which receives and supportsthe specimens to be embedded. A unique cassette permits the embedding ofa large number of tissue specimens simultaneously, thereby expeditingthe preparatory process and making the most efficient use of themachine. While manipulation of the tissue is not ordinarily required,means is provided to permit manipulation of the tissue within theindividual cuplike holders prior to or during embedding and while undervacuum. The manipulative means also includes additional metering meansto permit resin or other types of embedding substances other thanparaffin to be used and includes means to evacuate or degas theembedding resin prior to use. Several advantages are provided by thepresent design including single-step embedding, permitting the use ofirregular chunks or discs of paraffin. Embedding is performed undervacuum with degassed paraffin or resin. Conversion from the resin toparaffin option can be conveniently accomplished through replacement ofthe movable probe. The containers for embedding are relativelyinexpensive, permitting them to be discarded after each experiment toavoid any problems of contamination of future specimens. The samples maybe observed throughout the embedding step through the use of atransparent chamber which permits 360 observation. Breaking of thevacuum is easily accomplished and removal and setup for the nextembedding run is completed with unequal ease through the novel butsimple structural arrangement.

It is an object of this invention to provide a new and improved methodand apparatus for tissue preparation.

It is a further object of this invention to provide a new and improvedapparatus for tissue preparation of simplified design which permitsembedding of multiple samples without necessitating manipulation underordinary circumstances, however, means is provided to permitmanipulation should it become desirable.

It is a still further object of this invention to provide a new andimproved apparatus for tissue preparation having a novel supportarrangement having improved heat transfer characteristics which permitheating to melt the embedding material without terminating therefrigeration.

Objects in addition to those specifically set forth will become apparentupon reference to the accompanying drawings and following description.

In the drawings:

FIG. 1 is a fragmentary perspective view of the apparatus of the presentinvention having the resin option;

FIG. 2 is a top plan view of the cassette which is adapted to carry thecups supporting the tissue samples;

FIG. 3 is an enlarged cross-sectional view of the cassette and a cup foruse in the resin option and with a tissue sample in full elevation;

FIG. 4 is an enlarged view of a modified form of cup containing a tissuesample undergoing embedding in the paraffin option;

FIG. 5 is an enlarged cross-sectional view of the form shown in FIG. 1with the resin probe in position to feed resin to one of the tissueholding cups; and

FIG. 6 is a cross-sectional view similar to FIG. 3 illustrating theparaffin option.

Referring now to FIG. 1, the tissue drying and embedding accessory isindicated generally by the reference numeral 10 consisting of a closedchamber indicated generally at 11 and a resin metering assembly 12positioned on the chamber 11. The closed chamber 11 consists of atransparent cylinder or sleeve of plastic or the equivalent [5 which issealed with a vacuumtight fit along its lower margin to the top of afreeze dryer known in the art as Model No. 10-010 sold by The VirtisCompany, Inc. of Gardiner, N.Y. An O-ring slotted to receive the lowerend of the sleeve 15 is satisfactory to provide sealing with the top ofthe freeze dryer.

The transparent sleeve 15 is coaxially arranged with a stainless steelcondensing coil 13 helically wound in the manner indicated. The upperend of the transparent cylinder 15 is closed off by a transparent cover14 with a seal 19 interposed and serves to fluidtightly support theresin embedding assembly 12. The details of this arrangement will begiven in conjunction with the description of the enlarged views of FIGS.5 and 6.

As best seen in the enlarged fragmentary view of FIG. 5, the cover 14 isprovided with a shouldered central opening 16 which receives a sphere 17of nylon or the equivalent. The sphere is sealed for free movement inthe opening through an O-ring 18 which is supported on an axially facingportion or shoulder 20. A cylindrical bore 21 is formed through thecenter of the sphere l7 and receives the lower end of a glass tube 22forming a part of the resin-embedding assembly 12. The tube is sealedfor movement relative to the sphere through a pair of spaced O-ringshaving high-vacuum grease in between. The lower end of the tube 22 islocated within the chamber and the opposite end is integral with aconventional stopcock valve 23 which, in turn, is in communicationthrough a tube 24 with a resin reservoir 25. The upper end of thereservoir is connected to a flexible tube 27 through a stopcock valve 26with the opposite end of the flexible tube 27 in fluid communicationwith the chamber 15 through a three-way valve which is the subject of acopending patent application, Ser. No. 650,134, filed on June 29, 1967and now US. Pat. No. 3,509,909. The three-way valve 30 permits the resinreservoir 25 to be placed in communication with the drying chamber fordegassing of the resin prior to the embedding procedure and also permitsthe vacuum to be broken for ease in removing the cover.

The condenser coil 13 receives a sleeve 31 with a tight fit to providefor good thermal conductance and mechanical support. The sleeve 31supports a cup-shaped member 32 having a thickened base portion 33 andupstanding sidewalls 34. The sleeve 31 may be joined through the cup 32by any suitable means which will provide good heat transfer. The bottomof the cup 32 is provided with an opening 35 which receives a heatingelement 36 having a conducting wire 37 joined to a suitable plug 38which is mounted in the wall of the transparent sleeve 15 with avacuum-tight fit. The cup 32 supports a cassette 40 which receives aplurality of flexible tissue cups 41 (only two shown) which are manuallypressed into the eassette plate. The cups may be formed of plastic orthe equivalent and are readily available commercially.

As seen in FIG. 2, a typical cassette may consist of a flat disc havinga plurality of openings 43 which are sized so as to receive the cup 41which is shown in enlarged cross section in FIGS. 3 and 4. In the formof cup shown in FIG. 3, the tissue specimen 44 is supported intheplastic cup 41 which is pressfitted in any one of the 24 openings in theplate 40. Any number of openings may be provided in the plate 40,limited only by mechanical geometry or the capacity of the system. As isevident, a number of diverse samples may be dried simultaneously.

In FIG. 4, a modified form of tissue cup is shown at 45 having aplurality of perforations 46 in the bottom wall to allow wax or otherliquid embedding material 47 to flow into the cup from the bottom andenvelop the tissue specimen 48. This will be described in greater detailin connection with the paraffin option shown in FIG. 6.

In the paraffin option of the invention which is shown in enlarged crosssection in FIG. 6, the glass tube 22 may be replaced by a solid glassrod 50 which extends through the nylon sphere 17 and has a reduceddiameter at its end to form a manipulative probe 51. The rod 50 issealed to the central bore 21 of the nylon sphere 17 through the use ofO-rings held within the bore 21 through shoulders (not shown) formed atopposite ends of the bore 21. The area intermediate the rings 52 and 53is filled with a suitable sealing compound such as a high-vacuum greaseto assure a vacuumtight seal as well as relatively free movement of theprobe 50 relative to the sphere 17. From the foregoing, it is evidentthat conversion from the resin to paraffin option is accomplishedthrough substitution of the resin assembly for the manipulative probe.

In the paraffin option shown in FIG. 6, the container 32 is filled withchunks or blocks of paraffin which are heated to a temperaturesufficient to cause them to melt and form a pool as shown at 47 in FIG.6. Throughout the melting process, a

vacuum is drawn in the chamber to remove any entrained gas from theparaffin. Refrigeration is continuously applied and the heating means 36deenergized, causing the paraffin to solidify. When the paraffin 47reaches the solid state, the three-way valve 30 may be opened to admitair, permitting removal of the cover 14 and insertion of the cassette 40containing perforated cups 45. The cups 45 contain tissue specimenswhich may have been previously dried or, in the alternative, which havebeen prefrozen and may be dried within the tissue preparation chamber 15in advance of the embedding step. The cover 14 is replaced while therefrigeration is continuously applied. A vacuum is then applied to thechamber 15. The radiant heat transmitted through the walls of thetransparent chamber 12 and cover 14 is sufficient heat input to causesublimation of the tissue specimens to be embedded, causing the water tovaporize so the vacuum will cause its removal from the chamber. Whendrying is completed, the heater 36 is energized, causing the paraffin 47to liquefy. Throughout this step, the refrigeration is continued,however, the proximity of the heating element to the container issufficient to override the effect of the refrigeration and cause meltingof the parafiin 47.

In FIG. 4, the paraffin is in the liquid state and the cassette 40 andperforated cups 45 sink slowly into the liquid paraffin, allowing theparaffin to enter the cup 45 in the direction shown by the arrows andthereby envelop and permeate the dried specimen 48. If at any timeduring the process it is necessary to manipulate the tissue specimen,the probe 50 may be moved to any position to bring the end 51 intocontact with the specimen in any one of the cups to permit the same tobe oriented relative to the cup for best results in embedding.

After the specimen 48 is fully enveloped and embedded, the heater isdeenergized and the refrigeration becomes effective to causesolidification of the paraffin 47. The application of vacuum isdiscontinued, the valve 30 opened to vent the chamber and the cover isremoved. The cassette plate 40 with the specimens embedded in paraffinmay then be removed from the container for preservation and the studyprocedures outlined above. The process may be repeated as necessary.

In operation, the resin embedding option functions quite similarly tothe paraffin option, however, the embedding resin is metered into thecup under the influence of gravity. The tissue cup 41 of necessity mustbe of the form shown in FIGS. 3 and 5. The processing steps throughfreeze drying are the same as in the paraffin option, however, duringthe drying process the resin is degassed. To accomplish this, valve 26is opened as is the three-way valve 30 to apply vacuum to the resinreservoir 25. When the tissue is ready for embedding, valve 23 is openedafter the probe has been positioned over the cup 41 containing thetissue to be embedded. Theresin in the reservoir 25 flows under theinfluence of gravity into the cup and envelops the tissue specimen 44.The valve 23 is closed when the cup is filled and the resin probe 22moved from cup to cup with each cup filled to a level above the tissuespecimen.

As shown in dotted lines and more particularly described in the patenton the three-way valve 30 alluded to above, the vacuum may be broken byrotating the handle 60 to a position to admit air through the end 61permitting the cover 14 to be removed to remove the specimens. The useof the isolated cooling coil 13 as the sole support of the sleeve 31reduces the mechanical vibrations transmitted to the cassette 40 andtissue receiving cups 41 or 45. This damping of the vibrations isimportant to prevent mechanical abrasion of the tissue samples whilethey are being sublimated and otherwise unsupported, since it isconceivable that excessive vibrations could reduce these tissue samplesto powder if operation at a resonant frequency occurred.

It will be appreciated from a consideration of the foregoing that thefreeze drying and embedding accessory provides great versatility forexisting freeze dryers. The simplified chamber and embedding procedureof the present invention permits the use of chunks of paraffin asopposed to predimensioned discs. Moreover, conversion of the system fromparaffin to resin is easily accomplished through substitution of themanipulative probe 50 with the resin assembly 12. Multiple embedmentscan be made simultaneously and the process observed continuously througha full 360 due to the transparent chamber. Moreover, the use of atransparent chamber functions to provide sufficient radiant energy toeliminate the need for a controlled heat input during the freeze dryingprocess. The unique location of the heating means permits the heatingstep to be performed without cutting off the refrigeration, therebyexpediting the entire embedding process.

A further important aspect of this invention is that in either of theparaffin or resin options, the tissue embedding process is carried onwith the reservoir or cup-shaped member in direct metallic contact withthe condenser coil. In other words, there is no discontinuity betweenthe heated reservoir and the condenser coil. Nevertheless, the condensertemperature will be maintained, even though the reservoir is sometimesat a temperature of approximately C. since the heat transferred islimited by the thin wall sleeve 31 which connects the reservoir to thecoil. This sleeve provides a relatively slow heat transfer path so thatwhat heat does get through to the coils can be dissipated within thecapacity of the refrigeration system. This unique structure permits thecontinuous operation of the refrigeration system.

Upon a consideration of the foregoing, it will become obvious to thoseskilled in the art that various modifications may be made withoutdeparting from the invention embodied herein. Therefore, only suchlimitations should be imposed as are indicated by the spirit and scopeof the appended claims.

lclaim:

l. A tissue dryer for drying and embedding tissue samples and the likecomprising a cooling coil, support means including an axially extendingsleeve disposed within and contiguous to said cooling coil, in directheat transfer relationship with said cooling coil said support meansbeing mounted on said cooling coil, and supporting container means andmeans to elevate the temperature thereof to permit heating of embeddingmaterial, a cassette mounted on said support means having a plurality oftissue receiving cups thereon, closure means enveloping said supportmeans and cooling coil in a vacuumtight manner, and means to evacuatesaid closure means.

2. A tissue dryer for drying and embedding tissue samples and the likecomprising a cooling coil, support means disposed in heat transferrelationship with said cooling coil, a cassette mounted on said supportmeans and having a plurality of tissue-receiving cups thereon, saidsupport means including a reservoir, and each of said tissue receivingcups being perforated at its lower extremity to admit embeddingmaterial, said reservoir being filled with embedding material, andheating means to soften said embedding material to permit flow into saidtissue receiving cups, closure means enveloping said support means andcooling coil in a vacuumtight manner, and means to evacuate said closuremeans.

3. A tissue dryer for drying and embedding tissue samples and the likecomprising a cooling coil, support means disposed in heat transferrelationship with said cooling coil, a cassette mounted on said supportmeans and having a plurality of tissue receiving cups thereon, saidcooling coil being the sole support for said support means thereby toreduce the mechanical vibrations transmitted to said cassette and saidtissue receiving cups, closure means enveloping said support means andcooling coil in a vacuumtight manner, and means to evacuate said closuremeans.

4. A tissue dryer for drying and embedding tissue samples and the likecomprising a cooling coil, support means disposed in heat transferrelationship with said cooling coil, a cassette mounted on said supportmeans and having a plurality of tissue-receiving cups thereon, closuremeans enveloping said support means and cooling coil in a vacuumtightmanner, and means to evacuate said closure means, said closure meansincluding means to permit manipulation of the tissue in said tissuereceiving cups during drying thereof without breaking the vacuum in saidclosure.

5. The tissue dryer of claim 4 wherein said means to manipulate saidtissue in said cup includes means to supply embedding resin under vacuumto each of said cups.

1. A tissue dryer for drying and embedding tissue samples and the likecomprising a cooling coil, support means including an axially extendingsleeve disposed within and contiguous to said cooling coil, in directheat transfer relationship with said cooling coil said support meansbeing mounted on said cooling coil, and supporting container means andmeans to elevate the temperature thereof to permit heating of embeddingmaterial, a cassette mounted on said support means having a plurality oftissue receiving cups thereon, closure means enveloping said supportmeans and cooling coil in a vacuumtight manner, and means to evacuatesaid closure means.
 2. A tissue dryer for drying and embedding tissuesamples and the like comprising a cooling coil, support means disposedin heat transfer relationship with said cooling coil, a cassette mountedon said support means and having a plurality of tissue-receiving cupsthereon, said support means including a reservoir, and each of saidtissue receiving cups being perforated at its lower extremity to admitembedding material, said reservoir being filled with embedding material,and heating means to soften said embedding material to permit flow intosaid tissue receiving cups, closure means enveloping said support meansand cooling coil in a vacuumtight manner, and means to evacuate saidclosure means.
 3. A tissue dryer for drying and embedding tissue samplesand the like comprising a cooling coil, support means disposed in heattransfer relationship with said cooling coil, a cassette mounted on saidsupport means and having a plurality of tissue receiving cups thereon,said cooling coil being the sole support for said support means therebyto reduCe the mechanical vibrations transmitted to said cassette andsaid tissue receiving cups, closure means enveloping said support meansand cooling coil in a vacuumtight manner, and means to evacuate saidclosure means.
 4. A tissue dryer for drying and embedding tissue samplesand the like comprising a cooling coil, support means disposed in heattransfer relationship with said cooling coil, a cassette mounted on saidsupport means and having a plurality of tissue-receiving cups thereon,closure means enveloping said support means and cooling coil in avacuumtight manner, and means to evacuate said closure means, saidclosure means including means to permit manipulation of the tissue insaid tissue receiving cups during drying thereof without breaking thevacuum in said closure.
 5. The tissue dryer of claim 4 wherein saidmeans to manipulate said tissue in said cup includes means to supplyembedding resin under vacuum to each of said cups.